Inhibition of protein binding to mast cells

ABSTRACT

The invention relates to a compound inhibiting the binding of the free light chain of immunoglobin (Ig LC) to mast cells. It has been found that Ig LC is the agent responsible for the sensitization of mast cells. The compounds according to the invention can thus be used for the preparation of a drug for the treatment of a disease whose symptom is an elevated Ig LC concentration in serum or spinal fluid. The invention also relates to a method of screening a series of compounds on their ability to reduce the sensitization of mast cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a continuation of,International Application No. PCT/NL99/00430, filed on Jul. 7, 1999,designating the United States of America, the contents of which areincorporated by this reference, the PCT International Patent Applicationitself claiming priority from the Netherlands Application Serial No.1009601 filed Jul. 9, 1998.

TECHNICAL FIELD

[0002] The current invention relates to the field of immunology, andmore specifically, to components which sensitize mast cells.

BACKGROUND OF THE INVENTION

[0003] In the field of immunology, Askenase, P. W. et al (J. Exp. Med.157, p. 862-873 (1983)) describe a “T-cell factor” which sensitizes mastcells. This T-cell factor is a very impure composition. Until now, itwas not known which component/components was/were responsible for thesensitization.

DISCLOSURE OF THE INVENTIONI

[0004] Applicant has found that the free light chain of immunoglobulin(Ig LC) is a constituent of the T-cell factor and is the agentresponsible for the sensitization of mast cells. It has been found thatIg LC sensitizes the mast cells antigen-specifically.

[0005] Therefore the present invention relates to a compound whichinhibits the binding of the free light chain of immunoglobulin to mastcells, wherein the compound, in the presence of an equimolar quantity offree light chain (LC) of immunoglobulin reduces its binding by at least5%, said compound not being Tamm-Horsefall glycoprotein (THP), orLC-binding peptide fragments thereof.

[0006] Such a compound is of major importance for use as a means forsuppressing the unpleasant effects resulting from sensitizationexperienced by a patient. The compounds can be detected in a simplemanner, for example, by incubating a compound to be tested, togetherwith fluorescent labeled Ig LC and mast cells. With the aid of afluorescence microscope or, for quantitative measurement, a FluorescenceActivated Cell Sorter (FACA), inhibition may be assessed. Thisinhibition may occur due to competition between the compound and Ig LCfor binding to the mast cell.

[0007] According to a preferred embodiment, the compound can bind to thefree light chain of immunoglobulin, while the compound is capable ofcompeting with a peptide binding to the free light chain having theamino acid sequence (AHWSGHCCL) SEQ ID NO: 1 and that in the presence ofan equimolar quantity of the peptide, the compound reduces its bindingby at least 5%.

[0008] Huang Z. -Q. et al. (ref. 2) describe a unique urinary protein,Tamm-Horsefall glycoprotein (THP, also known as uromoduline) causingaggregation of immunoglobulin light chains and THP. This aggregatecauses renal failure due to clogging of the distal nephron of thekidney. The publication discloses tryptic peptides of THP also causingaggregation. The use thereof or THP as a drug is not disclosed, nor isthe binding thereof to mast cells disclosed.

[0009] Preferably, the compound reduces the binding of the peptide by atleast 10%, preferably by at least 25%, more preferably by at least 50%,even more preferably by at least 75% and most preferably by at least90%.

[0010] In principle, such compounds are very useful as an activecomponent for a pharmaceutical composition, particularly if binding isreduced by more than 50%.

[0011] Within the scope of the present invention, the peptide may alsobe used as the active component. It is also possible to use peptideswith unusual and/or modified amino acids. According to a preferredembodiment, the compound is a peptidomimeticum.

[0012] A suitable peptidomimeticum is, for example, a peptoid such as apeptoid corresponding with the peptide, but in which the side chains arelocated on the nitrogen atoms of the peptide backbone. In comparisonwith the original peptide, such a peptoid has a longer half-life in theblood. The synthesis of peptoids is well-documented in the art. The mostimportant difference with the synthesis of peptides is the differentstarting materials corresponding to the amino acids.

[0013] The present invention also relates to a method of screening aseries of compounds for their capability to bind the free light chain ofimmunoglobulin using a labeled compound capable of binding the freelight chain of immunoglobulin, and capable of competing with the peptidewith the amino acid sequence (AHWSGHCCL) of the formula sheet, whereinthe screening is performed using a test comprising a competitionreaction between the compound to be tested and the labelled compound.The test is suitably a homogenous test, making it possible to quicklyscreen compounds and to select active compounds.

[0014] In the present invention a homogenous test is understood to be atest wherein for detection it is not necessary to separate anon-complexed labelled peptide (or peptidomimeticum) from acomplexed-labelled peptide. Instead of the labelled peptide with theamino acid sequence (AHWSGHCCL) it is, of course, also possible to use acompound found with that peptide.

[0015] Two examples of very suitable homogenous tests are based onfluorescence (de)polarization or internal energy transfer respectively,as these allow for optimal use of respectively the difference in sizebetween complex and labelled peptide, and the small distance between thefluorophore and chromophore.

[0016] The present invention also relates to a method of screening aseries of compounds for their capability of reducing the sensitizationof mast cells, wherein the screening is performed by incubating acompound to be tested and a labelled free light chain of immunoglobulinwith a mast cell, and detecting reduced binding of the labelled freelight chain of immunoglobulin.

[0017] In cases like this it is preferred that the screening occursunder physiological conditions, as the compound will have to be activewhen used as a drug under those conditions.

[0018] It goes without saying that the compound may be used forpharmaceutical purposes, especially if the compound is pharmaceuticallyacceptable.

[0019] Thus the present invention also relates to an application of acompound (obtained) according to the present invention or Tamm-Horsfallglycoprotein (THP) or LC-binding peptide fragments thereof for thepreparation of a drug for a disease having as a symptom i) aconcentration of the free light chain of immunoglobulin in serum of atleast 8 mg/l, in particular of at least 15 mg/l and more in particular20 mg/l; and/or ii) a concentration of the free light kappa-chain ofimmunoglobulin in spinal fluid of at least 70 μg/l, in particular atleast 100 μg/l, and more in particular 150 μg/l; and/or iii) aconcentration of the free lambda-chain of immunoglobulin in spinal fluidof at least 300 μg/l, in particular at least 400 μg/l, and more inparticular 500 μg/l.

[0020] Important examples of such diseases are asthma, allergy,including contact allergy and occupational allergy, chronic inflammatorybowel disorders, viral infection and multiple sclerosis. Applicantconsiders the possibility that migraine is also included in the list ofdisorders.

[0021] According to an advantageous embodiment, a compound is used whichis a peptide or peptidomimeticum with a mass of less than 10 kDal,preferably less than 2 kDal.

[0022] If the peptide does not need to be synthesized because it isderived from a protein, the mass is of less importance, although thepeptide is then preferably non-immunogenic.

[0023] Thus the present invention also relates to a pharmaceuticalcomposition comprising a compound according to the invention orTamm-Horsfall glycoprotein (THP) or LC-binding peptides thereof togetherwith a pharmaceutically acceptable carrier or excipient.

[0024] Finally, the invention relates to a method of diagnosing adisease in a patient having an elevated level of the free light chain ofimmunoglobulin in a bodily fluid, wherein a foreign antigen specific forthe disease is contacted with the bodily fluid from the patient, andsubsequently the presence is determined of a complex of the foreignantigen and the free light chain of immunoglobulin.

[0025] The bodily fluid is suitably urine, serum or plasma. Spinalfluid, lung washing and sputum are considered as well. Thus, in thecontext of the present invention, a bodily fluid also comprises liquidsprepared 0006on the basis of the bodily material. The presence of acomplex can be detected by using one of many methods known in the stateof the art such as, for example, a sandwich ELISA wherein the complex isdetected using a labeled antibody directed against the free light chain.

[0026] Such a labeled antibody is suitably directed against a conservedpart of the free light chain.

DETAILED DESCRIPTION OF THE DRAWINGS

[0027] The present invention will now be illustrated by the followingexamples with reference to the drawings, wherein:

[0028]FIG. 1 represents a graph of the relative fluorescence as ameasure for the amount of bound Ig LC against the number of mast cells;

[0029]FIG. 2, portions A and B, show Western blots afterSDS-PAGEelectrophores is; and

[0030]FIG. 3 shows a graph for an ELISA-based binding assay.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Preparation 1

[0032] Preparation of Ig LC-binding Peptide LCBP.

[0033] The peptide Ac-AHWSGHCCL-NH₂ (SEQ ID NO: 1) was prepared using a430A Applied Biosystems Instruments (Foster City, Calif., U.S.A.) usingsolid-phase FastMoc chemistry. For the preparation a Tentagel-S-RAMresin was used as carrier material. Sensitive side chains were protectedusing His(Trt), Cys(TrT), Trp(boc), Ser(tBu). The peptide was releasedfrom the resin and the protective groups were removed using a mixture oftrifluoroacetic acid, ethane dithiol and water (95:2.5:2.5 v/v). The rawpeptide was precipitated using ether and purified by means ofpreparative HPILC. The purity of LCBP was verified using analytical HPLCand mass spectrometry.

[0034] Preparation 2

[0035] Isolation of Lymphocyte Factor

[0036] BALB/c mice (RIVM, Bilthoven, the Netherlands) wereskin-sensitized using picrylchloride (PLC), dinitrofluorobenzene oroxazolone as described earlier (ref. 1). Four days after sensitizationspleen cells (10×10⁶ cells/ml) were cultured for 24-48 hours in RPMImedium supplemented with penicillin, streptomycin and gentamycin. Thesupernatant was harvested and antigen-binding proteins were isolatedusing hapten-affinity chromatography (bovine gamma globulin or BSAprovided with hapten immobilized to Affigel-10 (Bio Rad I,abs.,Veenendaaj., the Netherlands)) as described by Ferguson. T. A. et al.(ref. 3) After washing the column with PBS+0.5 M NaCl, the proteins wereeluted with 5 ml 5 M guanidine solution. Subsequently, extensivedialysis against PBS took place. Proteins of biologically activesamples, such as determined with an ear swelling test (see hereinafter),were fractionated using 15% Tricine SDS-PAGE, blotted onto PVDF andsubsequently subjected to an Edman degradation for amino acid sequenceanalysis.

[0037] To determine the presence of kappa Ig LC, the hapten-bindingproteins were fractionated using 12.5% SDS-PAGE, blotted onto PVDF andtested with horseradish peroxidase-labeled anti-Ig kappa LC (The BindingSite, Birmingham, U.K.) in a dilution of 1:2000. Immunoreactive proteinswere visualized using ECL (Amersham Pharmacia Biotech Benelux,Roosendaal, the Netherlands) according to the manufacturer'srecommendations (FIG. 2, portion A). This showed that in lymphocytefactors specific for picric acid, dinitrofluorobenzene and exazolonrespectively, the presence of Ic LC could be demonstrated using ananti-kappa Ig LC-specific antibody.

[0038]FIG. 2, portion B shows that lymphocyte factor comprises a largevariety of antigen-binding proteins. The lanes labeled A (eluted with0.2 MN Na2CO₃) and B (void volume of column) are two fractions obtainedusing affinity chromatography. Of these fractions only fraction Aexhibited the biological activity demonstrated in Example 3. Of theprotein with an apparent molecular weight of 27 kDal (p27)(DIQMTQSPPSLSAXLG)(SEQ ID NO: 2) the N-terminal amino acid sequence wasdetermined, which corresponded to the sequence of Ig LC(DIQMTQSPSSLSASLG)(SEQ ID NO: 3) known from the literature.

EXAMPLE 1 Ig LC-binding to Mast Cells

[0039] Basophilic leukemia cells RBL-2H3 (a gift of C. Fewtrell. Ithaca,N.Y., U.S.A.) of the rat, an established model for mast cells, wereincubated with 200 ng/10⁵ cells Ig LC labeled with fluoresceinisothiocyanate. They were incubated for 30 minutes at 4° C. in thepresence or absence of 250 μg/ml of the peptide LCBP prepared inpreparation 1 (peptide binding to the light chain). Subsequently theywere washed using a phosphate-buffered saline supplemented with 1% v/vfoetal calf serum and 0.01% w/v sodium azide. Binding of FITC-labelledIg LC to RBL-2H3 cells was analyzed using a FACScan flow cytometer.

[0040] The curve 1 of FIG. 1 shows that the free light chain of Ig bindsto mast cells. Secondly, curves 2 and 3 of FIG. 1 show that this bindingcan be inhibited using LCBP (0.25 mg/ml and 0.50 mg/ml respectively).Curve 4 represents the autofluorescence of unlabelled RBL-2H3 cells.

EXAMPLE 2 Effect of Peptide of LCBP to the Airduct Response

[0041] 2.1 Sensitization of Mice

[0042] Lightly anaesthetized with halothane, mice were passivelysensitized by injection with trinitrophenyl (TNP)-specific Ig LC (2 μgin 50 μl of sterile saline) in the retroorbital plexus. Control micereceived only 50 μl of sterile saline. Thirty min. after injection,while being lightly anaesthetized with halothane, all mice receivedintranasally 50 μl PSA-solution (picrylsulphonic acid dissolved inphosphate-buffered saline).

[0043] 2.2 Effect of Ig LC and LCBP

[0044] A part of each of these two groups of mice simultaneouslyreceived 200 μg LCBP (the peptide prepared in Example 1) intranasally.

[0045] Bronchoconstriction was measured as described by Kraneveld A. D.et al. (ref. 3) and Zuany-Amorim et al. (ref. 6). In short, 5 minutesbefore intranasal application of PSA, mice were placed in aplethysmographic chamber (Buxco Electronics Inc., Shanon, Conn.) inorder to analyze respiration and to obtain basal line readings. Afterthe intranasal administration the animals were directly returned to thechamber. The respiratory resistance was measured for a period of 45minutes. The respiratory resistance is expressed as a dimensionlessvalue calculated by using the formula for the Penh (ref. 4). For eachmouse the maximum Penh values were measured during an interval of 1minute at the moments shown in Table 1. TABLE 1 PBS/PBS/ PBS/IgLCBP/PBS/ time PSA LC/PSA PSA LCBP/Ig LC/PSA (min.) (Penh) (Penh) (Penh)(Penh)   0¹ 0,62 ± 0,09 0,35 ± 0,03 0,44 ± 0,05 0,43 ± 0,05 2,5  0,60 ±0,20 1,98 ± 0,16 0,60 ± 0,12 0,87 ± 0,20  5 0,62 ± 0,20 2,29 ± 0,51 0,65± 0,23 1,12 ± 0,20 7,5  0,70 ± 0,30 5,29 ± 1,00 0,74 ± 0,12 0,87 ± 0,1010 0,60 ± 0,20 6,05 ± 1,90 0,53 ± 0,03 0,87 ± 0,20 15 0,70 ± 0,05 3,76 ±0,70 0,49 ± 0,06 0,85 ± 0,01 20 0,73 ± 0,20 1,88 ± 0,30 0,49 ± 0,03 0,52± 0,05

[0046] This experiment shows that intranasal administration of LCBPduring the passive sensitization (i.v.) with Ig LC can completelyinhibit the bronchoconstriction (elevation of Penh) induced by antigen(PSA).

EXAMPLE 3 Effect of Passive Sensitization with Ig LC on Ear Swelling.

[0047] Mice were, as described by Example 2.1, passively sensitized byinjection with a lymphocyte factor PLC-F obtained from a mousesensitized with picrylchloride or Ig LC specific for trinitrophenyl.Control mice received either only PBS or TNP-specific Ig HC (heavy chainof immunoglobulin). Thirty minutes after injection picrylchloride (50 μl0.8% picrylchloride (PCL) dissolved in olive oil) was applied to theear. After 2 hours, the thickness of the ear was measured (Table 2).TABLE 2 Increase in ear thickness Treatment (× 10⁻⁵ m) PCL-F 3,94 ± 0,56TNP-specific Ig LC 3,77 ± 0,46 PBS* 0,37 ± 0,40 TNP-specific Ig HC* 0,01± 0,32

[0048] This experiment shows that TNP-specific Ig LC has the same effectas lymphocyte factor. The heavy chain does not show this effect.

EXAMPLE 4 Bronchoconstriction with Mast-cell Deficient Mice

[0049] Mast cell-deficient mice (WBB6F1 W/Wv) (Jackson Labs, Bar Harbor,Me., USA belong to a strain of mice lacking mast cells. Their responseto challenge with picryl sulphonic acid 30 minutes after sensitisationwith PBS (vehicle), LC and IgE (5 microgram in 50 microliter each) wascompared with the response of strain WBB6F1 +/+ (Jackson Labs, BarHarbor, Me., USA) mice having a similar genetic make-up but notmast-cell deficient. Picryl sulphonic acid (50 microliter, 0,6% (w/v))was administered intranasaly and the Penh values were obtained asdescribed in Example 2. The results are given in Table 3. TABLE 3 TimePBS LC IgE (min.) mean mean mean Mast cell-deficient animals 0 0.88 (0,11) 0,72  (0,10) 0,84  (0,10) 2,5 1,15  (0,44) 0,82  (0,23) 1,84 (0,60) 5 1,15  (0,17) 1,01  (0,16) 1,96  (0,48) 7,5 1,37  (0,35) 1,24 (0,41) 2,51  (0,68) 10 1,22  (0,36) 1,07  (0,18) 2,02  (0,89) 15 1,22 (0,16) 1,10  (0,28) 1,64  (0,29) 20 0,82  (0,12) 0,85  (0,19) 1,21 (0,26) control 0 0,72  (0,04) 0,98  (0,12) 0,95  (0,21) 2,5 1,02 (0,22) 2,46  (0,51) 2,17  (0,44) 5 1,25  (0,22) 6,14  (1,87) 4,34 (1,97) 7,5 1,05  (0,23) 8,17  (1,10) 4,73  (1,32) 10 1,33  (0,15) 8,89 (1,95) 6,83  (1,22) 15 1,17  (0,19) 4,41  (1,30) 3,97  (0,86) 20 0,98 (0,20) 2,58  (0,65) 2,04  (0,43)

[0050] The standard error (SE) is shown between parenthesis

[0051] From this table it can be seen that the mast-cell deficient miceare not sensitized by LC.

EXAMPLE 5 ELISA-based Binding Assay

[0052] Wells of a microtiter plate were coated at room temperatureovernight with 2 μg/ml immunoglobulin light chains or, as a firstcontrol, Bovine Serum Albumin (BSA). Also, as a second control, wellswere treated with 250 mM glycine buffer, pH=9.5. The wells were emptiedand washed 5 times with 0,05% Tween-20 in PBS. The wells were blockedwith HPE-buffer (High Performance Elisa buffer, CLB, Amsterdam, TheNetherlands) for 1 hour, and subsequently the wells were washed againwith 0,05% Tween-20 in PBS. Human uromodulin was diluted in HPE-bufferand incubated for 2 hours. The wells were washed 5 times with 0,05%;Tween-20 in PBS.

[0053] To detect bound uromodulin, 1/5000 diluted rabbit anti-humanuromodulin antiserum was added (Anawa Trading, Zürich, Switzerland) andincubated for 1 hour. After washing 5 times with PBS, 0.05% Tween20anti-rabbit-IgG conjugated to horse radish peroxidase (CLB) was addedand incubated for 1 hour. Bound peroxidase was detected as is well-knownin the art using 3,5,3′,5′-tetramethyl-benzidine/H₂O₂ in 0,11 M sodiumacetate pH 5,5. The reaction was stopped using an equal volume of 2 MH₂SO₄ and the absorbance was read at 450 nm. The data obtained aredepicted in FIG. 3 which shows that a uromodulin concentration within,for example, 4-40 μg/ml, is an excellent concentration for repeating theabove ELISA to detect novel compounds according to the presentinvention. To this end, uromodulin and the compound (preferably atseveral concentrations) to be investigated are incubated simultaneouslyin order to compete with each other.

1 3 1 9 PRT Unknown peptide 1 Ala His Trp Ser Gly His Cys Cys Leu 1 5 216 PRT Unknown protein with apparent molecular weight of 27kDal 2 AspIle Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Xaa Leu Gly 1 5 10 15 316 PRT Unknown N-terminal amino acid sequence 3 Asp Ile Gln Met Thr GlnSer Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15

What is claimed is:
 1. A compound that inhibits the binding of the free light chain of immunoglobulin to mast cells, wherein the compound, in the presence of an equimolar quantity of the free light chain of immunoglobulin, reduces binding between the free light chain of immunoglobulin and said mast cells by at least 5%, said compound not being Tamm-Horsefall glycoprotein (THP), or LC-binding peptide fragments thereof.
 2. The compound of claim 1, wherein the compound can bind to the free light chain of immunoglobulin and can compete with a peptide capable of binding to the free light chain of immunoglobulin, said peptide having the amino acid sequence (AHWSGHCCL)(SEQ ID NO:1), and wherein said compound, in the presence of an equimolar quantity of said peptide, reduces binding between said peptide and said free light chain of immunoglobulin by at least 5%.
 3. The compound of claim 1 or 2, wherein the compound reduces the binding between the peptide and the free light chain of immunoglobulin by at least 10%, preferably by at least 25%, more preferably by at least 50%, even more preferably by at least 75%, and most preferably by 90%.
 4. The compound of claim 2 or 3, wherein the compound is a peptidomimeticum.
 5. The compound according to any one of the preceding claims, wherein the compound is a pharmaceutically acceptable compound.
 6. A method of screening a series of compounds based on their ability to bind the free light chain of immunoglobulin, said method comprising: using a labeled compound capable of binding the free light chain of immunoglobulin and capable of competing with a peptide for binding to the free light chain of immunoglobulin; and performing a test comprising a competition reaction between at least one compound of said series compounds and said peptide for binding to the light chain of immunoglobulin.
 7. The method according to claim 6, wherein said test is a homogenous test.
 8. The method according to claim 6 or 7, wherein the test is based on fluorescence (de)polarization or internal energy transfer.
 9. A method for screening a series of compounds based on their ability to reduce the sensitization of mast cells, said method comprising: incubating at least one compound of said series of compounds with a labeled free light chain of immunoglobulin with said mast cells; and detecting reduced binding of the labeled free light chain of immunoglobulin to said mast cells.
 10. A compound for use in treating a disease, said disease characterized by symptoms comprising: i) a concentration of the free light chain of immunoglobulin in serum of at least 8 mg/l, in particular of at least 15 mg/l and more in particular 20 mg/l; and/or ii) a concentration of the free light kappa-chain of immunoglobulin in spinal fluid of at least 70 μg/l, in particular at least 100 μg/l, and more in particular 150 μg/l; and/or iii) a concentration of the free lambda-chain of immunoglobulin in spinal fluid of at least 300 μg/l, in particular at least 400 μg/l, and more in particular 500 μg/l, said drug comprising a compound according to any one of the claims 1 to 5, the compound obtained by using the method according to any one of the claims 6 to 9, Tamm-Horsefall glycoprotein (THP) and LC-binding peptide fragments thereof.
 11. The drug of claim 10, wherein the compound is a peptide or peptidomimeticum with a mass of less than 10 kDal, preferably less than 2 kDal.
 12. The drug of claim 10 or 11, wherein the disease is selected from the group consisting of asthma, allergy, chronic inflammatory bowel disorders, viral infection and multiple sclerosis.
 13. A pharmaceutical composition comprising a compound according to any one of the claims 1 to 5 or obtained according to any one of the claims 6 to 9 or Tamm-Horsefall glycoprotein (THP) or LC-binding peptides thereof together with a pharmaceutically acceptable carrier or excipient.
 14. A method of diagnosing a disease in a patient having an elevated level of the free light chain of immunoglobulin in a bodily fluid, said method comprising: contacting a foreign antigen specific for the disease with the bodily fluid from the patient; and determining the presence of a complex of the foreign antigen and the free light chain of immunoglobulin.
 15. The method according to claim 14, wherein determining the complex comprises using a labeled antibody directed against the free light chain of immunoglobulin. 